Sampling devices and methods for concentrating microorganisms

ABSTRACT

The present disclosure describes methods for concentrating microorganisms with concentration agents in a sampling device and the sampling device described herein. More specifically, methods for concentrating microorganisms from large volume samples with concentration agents in a sampling device can provide for rapid, low cost, simple (involving no complex equipment or procedures), and/or effective processes under a variety of conditions.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.15/091,024, filed Apr. 5, 2016, which is a division of U.S. patentapplication Ser. No. 13/142,288, filed Sep. 21, 2011, now patented asU.S. Pat. No. 9,328,325, issued May 3, 2016, which is a national stagefiling under 35 U.S.C. 371 of PCT/US2009/069780, filed Dec. 30, 2009,which claims priority to U.S. Provisional Patent Application No.61/141,900, filed Dec. 31, 2008, the disclosures of which areincorporated by reference in their entirety herein.

FIELD

The present disclosure relates to sampling devices and methods forconcentrating microorganisms using such devices.

BACKGROUND

Food-borne illnesses and hospital-acquired infections resulting frommicroorganism contamination are a concern in numerous locations all overthe world. Thus, it is often desirable or necessary to assay for thepresence of bacteria or other microorganisms in various clinical, food,environmental, or other samples, in order to determine the identityand/or the quantity of the microorganisms present.

Bacterial DNA or bacterial RNA, for example, can be assayed to assessthe presence or absence of a particular bacterial species even in thepresence of other bacterial species. The ability to detect the presenceof a particular bacterium, however, depends, at least in part, on thenumber of the bacterium in the sample being analyzed. Bacterial samplescan be plated or cultured to increase the numbers of the bacteria in thesample to ensure an adequate level for detection, but the culturing stepoften requires substantial time and therefore can significantly delaythe assessment results.

Concentration of the bacteria in the sample can shorten the culturingtime or even eliminate the need for a culturing step. Thus, methods havebeen developed to isolate (and thereby concentrate) particular bacterialstrains by using antibodies specific to the strain (for example, in theform of antibody-coated magnetic or non-magnetic particles). Suchmethods, however, have tended to be expensive and still somewhat slowerthan desired for at least some diagnostic applications.

Concentration methods that are not strain-specific have also been used(for example, to obtain a more general assessment of the microorganismspresent in a sample). After concentration of a mixed population ofmicroorganisms, the presence of particular strains can be determined, ifdesired, by using strain-specific probes.

Non-specific concentration or capture of microorganisms has beenachieved through methods based upon carbohydrate and lectin proteininteractions. Chitosan-coated supports have been used as non-specificcapture devices, and substances (for example, carbohydrates, vitamins,iron-chelating compounds, and siderophores) that serve as nutrients formicroorganisms have also been described as being useful as ligands toprovide non-specific capture of microorganisms. Various inorganicmaterials (for example, hydroxyapatite and metal hydroxides) have beenused to non-specifically bind and concentrate bacteria.

Physical concentration methods (for example, filtration, chromatography,centrifugation, and gravitational settling) have also been utilized fornon-specific capture, with and/or without the use of inorganic bindingagents. Such non-specific concentration methods have varied in speed,cost (at least some requiring expensive equipment, materials, and/ortrained technicians), sample requirements (for example, sample natureand/or volume limitations), space requirements, ease of use (at leastsome requiring complicated multi-step processes), suitability foron-site use, and/or effectiveness.

SUMMARY

The present disclosure describes a method for concentratingmicroorganisms with a concentration agent in a sampling device. Morespecifically, methods for concentrating microorganisms from large volumesamples in the presence of concentration agents in such devices canprovide for rapid, low cost, simple (involving no complex equipment orprocedures), and/or effective processes for concentratingmicroorganisms.

In one aspect, a method is provided for concentrating microorganisms.The method includes providing a unitary sampling device comprising afirst reservoir, a second reservoir and a passageway having a firstopening in communication with the first reservoir and a second openingin communication with the second reservoir, wherein fluid can flowbetween the two reservoirs through the passageway. The entire volume ofthe first reservoir is above the first opening when the unitary samplingdevice is in an upright position, and the entire volume of the secondreservoir is not above the second opening when the unitary samplingdevice is in any position. The second reservoir has at least oneresealable external opening. The method includes mixing a concentrationagent and a sample comprising a microorganism in the unitary samplingdevice to provide a microorganism bound composition. The method alsoincludes inverting the unitary sampling device such that the secondreservoir is oriented substantially above the first reservoir forcollecting a major portion of the microorganism bound composition fromthe second reservoir.

In one aspect, a unitary sampling device is provided. The unitarysampling device comprises a first reservoir having a first opening, asecond reservoir having a second opening and at least one resealableexternal opening. The unitary sampling device also includes a passagewayconnecting the first reservoir to the second reservoir. The passagewayhaving the first opening is in communication with the first reservoirand the second opening is in communication with the second reservoir.The entire volume of the first reservoir is above the first opening whenthe unitary sampling device is in an upright position. The entire volumeof the second reservoir is not above the second opening when the unitarysampling device is in any position.

In one aspect, a method for concentrating microorganisms is provided.The method includes providing a dual component sampling devicecomprising a first reservoir having a main body, a resealable externalopening and a first end shaped so that it is narrower at the part mostdistal from the main body. The most distal part of the first end has afirst connector connected to a detachable aspirable second reservoirhaving a second connector. The first reservoir is oriented substantiallyabove the detachable aspirable second reservoir. The first connector iscapable of attaching to the second connector so that fluid can flowbetween the two reservoirs. The method includes mixing a concentrationagent and a sample comprising a microorganism in the dual componentsampling device to provide a microorganism bound composition. The methodalso includes directing the microorganism bound composition from thefirst end of the first reservoir into the detachable aspirable secondreservoir.

In one aspect, a dual component sampling device is described. The dualcomponent sampling device comprises a first reservoir having a mainbody, a resealable external opening and a first end shaped so that it isnarrowest at the part most distal from the main body. The most distalpart has a first connector. The dual component sampling device includesa detachable aspirable second reservoir having a second connector. Thefirst connector attaches to the second connector so that fluid can flowbetween the first reservoir and the detachable aspirable secondreservoir.

In one aspect, a method for concentrating microorganisms is provided.The method includes providing a unitary sampling device comprising afirst reservoir having a first opening and at least one resealableexternal opening, and a second reservoir having a second opening. Theunitary sampling device also includes an element comprising a housingand a movable feature residing within the housing, and a second externalopening. The movable feature has at least a first location and a secondlocation. The interior of the second reservoir is located within themovable feature and the second opening resides on the exterior or aportion of the movable feature. The first reservoir is located above theelement when the unitary sampling device is in an upright position. Thesecond external opening is located below the element when the unitarysampling device is in the upright position. At the first location, afirst passageway connects the first reservoir to the second reservoir sothat the first opening is in communication with the second opening. Atthe second location, a second passageway connects the second reservoirto the second external opening so that the second opening is in fluidcommunication with the second external opening. The method includesmixing a concentration agent and a sample comprising a microorganism inthe sampling device to provide a microorganism bound composition. Themethod also includes transferring the microorganism bound compositionfrom a first location to a second location so that the second opening isin fluid communication with the second external opening.

In one aspect, a unitary sampling device is provided. The unitarysampling device comprises a first reservoir having a first opening andat least one resealable external opening, a second reservoir having asecond opening, an element, and a second external opening. The elementcomprises a housing and a movable feature residing within the housing.The movable feature has at least a first location and a second location.The interior of the second reservoir is located within the movablefeature and the second opening resides on the exterior of a portion ofthe movable feature. The first reservoir is located above the elementwhen the unitary sampling device is in an upright position. The secondexternal opening is located below the element when the unitary samplingdevice is in the upright position. At the first location, the firstpassageway connects the first reservoir to the second reservoir so thatthe first opening is in fluid communication with the second opening. Atthe second location, the second passageway connects the second reservoirto the second external opening so that the second opening is in fluidcommunication with the second external opening.

In one aspect, a method for concentrating microorganism is provided. Themethod includes providing a unitary sampling device comprising a firstreservoir having a first opening, a first resealable external openingand a first volume. The unitary sampling device comprises a secondreservoir having a second opening, a second external opening and asecond volume. The unitary sampling device also comprises a plungerhaving a seal. The seal resides on a portion of the plunger proximate toa distal end of the plunger. The seal isolates the second volume of thesecond reservoir from the first volume of the first reservoir. Thesecond volume is removed through the second external opening, and thefirst reservoir is located above the second reservoir when the samplingdevice is in an upright position. The method includes mixing aconcentration agent and a sample comprising a microorganism in theunitary sampling device to provide a microorganism bound composition.The method also includes transferring the microorganism boundcomposition located in the second reservoir through the second externalopening with the plunger.

In one aspect, a unitary sampling device is provided. The unitarysampling device comprises a first reservoir, a second reservoir, and aplunger. The first reservoir has a first opening, a first resealableexternal opening and a first volume. The second reservoir has a secondopening, a second external opening and a second volume. The plunger hasa seal. The seal resides on a portion of the plunger proximate to adistal end of the plunger. The seal traps the second volume of thesecond reservoir from the first volume of the first reservoir. Thesecond volume is removed through the second external opening, and thefirst reservoir is located above the second reservoir when the samplingdevice is in an upright position.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a method for concentratingmicroorganisms.

FIG. 2 is a schematic representation of a unitary sampling device havinga first reservoir, a second reservoir and a passageway.

FIG. 3 is schematic representation of a dual component sampling devicehaving a first reservoir and a detachable aspirable second reservoir.

FIG. 4 is a schematic representation of a unitary sampling device ofFIG. 2 having a resealable external opening equipped with a closurehaving a protrusion extending into a passageway.

FIG. 5 is a schematic representation of a unitary sampling device ofFIG. 2 having a resealable external opening equipped with a closurehaving a protrusion not extending into the passageway.

FIG. 6 is a schematic representation of a unitary sampling device ofFIG. 2 having a passageway centered on an axis extending from the centerof the first reservoir to the center of the second reservoir.

FIG. 7 is a schematic representation of a unitary sampling device ofFIG. 2 having a passageway not centered on an axis extending from thecenter of the first reservoir to the center of the second reservoir.

FIG. 8 is a schematic representation of a unitary sampling device havinga first reservoir, a second reservoir, an element, and a second externalopening. The unitary sampling device illustrated at a first location.

FIG. 9 is a schematic representation of a unitary sampling device ofFIG. 8; the unitary sampling device illustrated at a second location.

FIG. 10 is a schematic representation of a side view of the element ofFIG. 8.

FIG. 11 is a schematic representation of a unitary sampling devicehaving a first reservoir, a second reservoir, and a plunger. The plungerpositioned above the second reservoir.

FIG. 12 is a schematic representation of the unitary sampling device ofFIG. 11 having the plunger extending through the second reservoir.

DETAILED DESCRIPTION

Although the present disclosure is herein described in terms of specificembodiments, it will be readily apparent to those skilled in the artthat various modifications, rearrangements, and substitutions can bemade without departing from the spirit of the invention. The scope ofthe present invention is thus only limited by the claims appendedherein.

The recitation of numerical ranges by endpoints includes all numberssubsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3,3.8, 4, and 5).

As included in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to acomposition containing “a compound” includes a mixture of two or morecompounds. As used in this specification and appended claims, the term“or” is generally employed in its sense including “and/or” unless thecontent clearly dictates otherwise.

Unless otherwise indicated, all numbers expressing quantities oringredients, measurement of properties and so forth used in thespecification and claims are to be understood as being modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the foregoingspecification and attached claims are approximations that can varydepending upon the desired properties sought to be obtained by thoseskilled in the art utilizing the teachings of the present disclosure. Atthe very least, each numerical parameter should at least be construed inlight of the number of reported significant digits and by applyingordinary rounding techniques. Notwithstanding that the numerical rangesand parameters setting forth the broad scope of the disclosure areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains errors necessarily resulting from the standarddeviations found in their respective testing measurements.

The present disclosure describes methods for concentratingmicroorganisms from large volume samples. A unitary sampling devicehaving a first reservoir and a second reservoir is provided for mixing aconcentration agent and a sample comprising a microorganism for forminga microorganism bound composition. A dual component sampling device isalso described. The concentration agent concentrates at least a portionof the microorganisms present in the sample. The microorganism boundcomposition is collected in a second reservoir of the sampling device.

Unitary sampling devices having an integrated (e.g., one piececonstruction) first reservoir and a second reservoir are provided.Similarly, dual component sampling devices described herein refer to atwo component device having an independent first reservoir and anindependent second reservoir (e.g., detachable first and secondreservoirs). The first reservoir and the second reservoir can beconnected to form the dual component sampling device. The firstreservoir of the sampling devices typically has a larger volume than thevolume of the second reservoir. The second reservoir is useful forcollecting smaller volume samples containing the microorganism boundcompositions.

Sampling devices described herein provide for rapid and efficientcollection of large volume samples having low microorganismconcentrations. In some examples, such sampling devices can be sealed,opened for containing a large volume sample, and then resealed forconcentrating microorganisms present in the samples. Concentrationagents are added to the sampling devices for mixing with the samples andforming microorganism bound compositions. The sampling devices of thepresent application described here are compact, portable (e.g., use infield environments), and disposable thus eliminating the possibility ofcontamination between samples.

Current techniques, for example, such as membrane filtration andcentrifugation, are used for obtaining a direct count of microorganismsfrom large volume samples. However, these methods for microbiologicalanalysis of samples such as water are generally expensive multistepprocedures, require sophisticated equipment, and highly trainedpersonnel. Additionally, operational requirements for membranefiltration or centrifugation techniques make these techniques difficultto perform on-site (e.g., in a field environment).

Methods for enumerating microorganism in water samples are described in,for example, “Standard Methods for the Examination of Water andWastewater (SMEWW), 21^(st) Edition, American Public Health Association,the American Water Works Association, and the Water EnvironmentFederation. Such methods and water quality testing regulations stipulatetesting 100 ml sample volumes which can not be easily performed in afield environment.

FIG. 1 is a process flow diagram for concentrating microorganism by themethods described herein. As illustrated in FIG. 1, a large volumesample comprising microorganisms is added to a sampling device. Thelarge volume sample is mixed with a concentration agent in the samplingdevice to form a microorganism bound composition. The microorganismbound composition in then collected in the second reservoir. In someembodiments, the concentration agent can be added to the sampling devicebefore the addition of the sample comprising microorganisms. In someembodiments, the concentration agent can be added to the sampling deviceafter the addition of the sample comprising microorganisms. In someembodiments, the sampling device can contain a concentration agent forefficient and portable use in field applications. At the site of sampleacquisition, the sampling device having the concentration agent can beopening for delivery and containment of the sample. After themicroorganism bound composition has been formed from mixing theconcentration agent and the sample, the microorganism bound compositioncan be collected for further analyses.

Each of the sampling devices described herein provide a first reservoirand a second reservoir. The first reservoir is designed to accommodate alarge volume sample having sufficient volume for mixing of the sampleand the concentration agent to form the microorganism bound composition.Microorganism bound compositions can be, for example, dispersible in thesample, and then collected or transferred from the first reservoir tothe second reservoir of the sampling device. The second reservoir has asmaller volume than the first reservoir in order to contain themicroorganism bound composition.

FIG. 2 illustrates a unitary sampling device 10. Unitary sampling device10 comprises a first reservoir 20, a second reservoir 35, a passageway40 and a resealable external opening 30. An opening 25 adjacent to thefirst reservoir 20 is optional. The first reservoir 20 is connected tothe second reservoir 35 at an interface 55 providing the unitarystructure. Both the first reservoir 20 and the second reservoir 35 havea volume. The first reservoir 20 has a first volume 60 and the secondreservoir 35 has a second volume 45. Passage way 40 has a first opening22 in communication with the first reservoir 20 and a second opening 27in communication with the second reservoir 35. The first reservoir 20contacts the second reservoir 35 at an interface 55.

In some embodiments, a sample comprising at least one microorganism canbe added to the unitary sampling device 10 of FIG. 2 via the resealableexternal opening 30. A concentration agent can be added to the samplewithin the unitary sampling device 10, or the concentration agent can bepresent in the unitary sampling device 10 prior to the addition of thesample. The sample and the concentration agent can be mixed in the firstreservoir 20 of the unitary sampling device 10 in an upright position,such that the entire volume of the first reservoir 20 is above the firstopening 22 to provide the microorganism bound composition. Themicroorganism bound composition can, for example, settle from the firstreservoir 20 through the passageway 40 into the second reservoir 35. Theunitary sampling device 10 can be inverted so that the second reservoiris oriented substantially above the first reservoir. At least a majorportion of the microorganism bound composition can be collected in thesecond volume 45 of the second reservoir 35. The unitary sampling device10 provides for efficient transfer of the microorganism boundcomposition from the first reservoir 20 to the second reservoir 35.

Dual component sampling device 300 is illustrated in FIG. 3. Dualcomponent sampling device 300 comprises a first reservoir 320 orientedsubstantially above a detachable aspirable second reservoir 340. Thefirst reservoir 320 has a main body 355, a resealable external opening315, a first end 325, a first volume 310, and a first connector 330. Thefirst connector 330 is attached to the detachable aspirable secondreservoir 340 at an intersection 335. The detachable aspirable secondreservoir 340 has a second volume 350 and a second connector 345 incontact with the first connector 330 of the first reservoir 320 residingat the intersection 335.

In some embodiments, a sample comprising a microorganism can be added tothe dual component sampling device 300 at the resealable externalopening 360 of FIG. 3. A concentration agent can be added to the dualcomponent sampling device 300 prior to or after the addition of thesample. The sample and the concentration agent can be mixed in the firstreservoir 320 to provide the microorganism bound composition. In someembodiments, the microorganism bound composition can travel through thesample within the main body 355 by gravity, for example, to the firstend 325 of the first reservoir 320. The first connector 330 of the firstreservoir 320 can be connected to the detachable aspirable secondreservoir 340 at the second connector 345 at intersection 335. Themicroorganism bound composition can be directed from the first end 325to the detachable aspirable second reservoir 340 via the conical shapeof the first end 325 of the first reservoir 320. A known volume of themicroorganism bound composition can be collected in the detachableaspirable second reservoir 340 for further analyses.

FIG. 4 illustrates one embodiment of a unitary sampling device 400. Thesecond reservoir 435 has a resealable external opening 450 comprising aclosure 430 having a protrusion 465. The protrusion 465 extends throughpassageway 440 from the second opening 470 to the first opening 445 whenthe closure 430 is attached to the resealable external opening 450. Theprotrusion 465 is selected to prevent the microorganism boundcompositions from re-entering first reservoir 480 after inverting theunitary sampling device 400 and collecting such microorganism boundcompositions in the second reservoir 435.

In some embodiments, the protrusion 465 can isolate the first reservoir480 from the second reservoir 435 prior to the addition of the sampleand/or the concentration agent. In some embodiments, the protrusion 465can be used to isolate a least a portion of a microorganism boundcomposition within the second reservoir 435 from the first reservoir480. The protrusion 465 can reduce or prevent the microorganism boundcomposition from flowing through the first opening 445 via thepassageway 440 and through the second opening 470 of the unitarysampling device 400.

FIG. 5 illustrates one embodiment of a unitary sampling device 500. Thesecond reservoir 535 has a resealable external opening 550 comprising aclosure 530 having a protrusion 565. The protrusion 565 extends from theclosure 530 in proximity of the second opening 570 without extendinginto the passageway 540 when the closure 530 is attached to theresealable external opening 550. The protrusion 565 is selected to atleast partially block microorganism bound compositions from re-enteringfirst reservoir 580 after inverting the unitary sampling device 500 andcollecting the microorganism bound compositions in the second reservoir535.

In some embodiments, the protrusion 565 can partially isolate the firstreservoir 580 from the second reservoir 535. After mixing theconcentration agent and a sample comprising a microorganism in theunitary sampling device 500, the unitary sampling device 500 can beinverted so that the second reservoir 535 is oriented substantiallyabove the first reservoir 580. During the inverting step, the protrusion565 of the closure 530 can direct at least a portion of themicroorganism bound composition into the second reservoir 535 forcollection.

FIG. 6 illustrates one embodiment of a unitary sampling device 610. Thepassageway 620 of the unitary sampling device 610 comprises a firstopening 615 and a second opening 625. The passageway 620 is centered onan axis 605 extending from the center of the first reservoir 630 to thesecond reservoir 640.

In some embodiments, passageway 620 of unitary sampling device 610 ofFIG. 6 can provide for flow of a microorganism bound composition fromthe first reservoir 630 at the first opening 615 through passageway 620to the second opening 625 of the second reservoir 640. The unitarysampling device 610 can be inverted so that the second reservoir 640 isoriented above the first reservoir 630 so that at least a major portionof the microorganism bound composition can collect in the secondreservoir 640.

FIG. 7 illustrates one embodiment of a unitary sampling device 710. Thepassageway 740 of the unitary sampling device 710 comprises a firstopening 715 and a second opening 725. Passageway 740 is not centered onaxis 705 extending from the center of the first reservoir 725 to thesecond reservoir 735.

In some embodiments, passageway 740 of unitary sampling device 710 ofFIG. 7 can provide for flow of a microorganism bound composition fromthe first reservoir 725 through passageway 740 being not centered (e.g.,off-centered) on axis 705. The unitary sampling device 710 can beinverted so that the second reservoir 735 is oriented above the firstreservoir 725 so that at least a major portion of the microorganismbound composition can collect in the second reservoir 735.

FIG. 8 illustrates a unitary sampling device 800. Unitary samplingdevice 800 comprises a first reservoir 810, a second reservoir 850, anelement 820, and a second external opening 880. The first reservoir 810has a first opening 860 and at least one resealable external opening890. The second reservoir 850 has a second opening 870. The element 820comprises a housing 830 and a movable feature 840 residing within thehousing 830. The movable feature 840 has a first location where a firstpassageway connects the first opening 860 to the second opening 870 suchthat second reservoir 850 is in fluid communication with the firstreservoir 810. The interior of the second reservoir 850 is locatedwithin the movable feature 840 and the second opening 870 resides on theexterior of a portion of the movable feature 840. The first reservoir810 is located above the element 820 when the unitary sampling device800 is in an upright position.

In some embodiments, the second reservoir 850 of unitary sampling device800 can have a first location for containing at least a portion of amicroorganism bound composition. The second reservoir 850 typically hasa fixed volume to collect the microorganism bound composition. Themovable feature 840 containing the second reservoir 850 can be rotatedto select a location between the first reservoir 810 and the secondreservoir 850.

FIG. 9 illustrates a unitary sampling device 900 comprising a firstreservoir 910, a second reservoir 950, an element 920 and a secondexternal opening 980. The first reservoir 910 has a first opening 960and at least one resealable external opening 990. The second reservoir950 has a second opening 970. The element 920 comprises a housing 930and a movable feature 940 residing within the housing 930. The movablefeature 940 has a second location where a second passageway connects thesecond reservoir 950 to the second external opening 980 so that thesecond opening 970 is in fluid communication with the second externalopening 980.

In some embodiments, the second reservoir 950 residing within themovable feature 940 of the unitary sampling device 900 can have a secondlocation so that a fixed volume of the microorganism bound compositionretained within the second reservoir 950 can be transferred via a secondpassageway. The second passageway at the second location connects thesecond reservoir 950 to the second external opening 980 for delivery ofthe microorganism bound composition from the second reservoir 950.

FIG. 10 illustrates a side view of element 1020. Element 1020 has ahousing 1030 and a movable feature 1040. Movable feature 1040 has afirst component 1070 for moving the second reservoir from a firstlocation to a second location (not shown). In some embodiments, thefirst component 1070 protrudes from the movable feature 1040 foraccessability to the movable feature 1040.

FIG. 11 illustrates a unitary sampling device 1100. Unitary samplingdevice 1100 comprises a first reservoir 1110, a second reservoir 1120,and a plunger 1130. The first reservoir 1110 has a first opening 1190, afirst resealable external opening 1170, and a first volume 1115. Thesecond reservoir 1120 has a second external opening 1140, and a secondvolume 1125. The plunger 1130 has a seal 1160 residing on a portion ofthe plunger 1130 proximate to a distal end 1155. The seal 1160 isolatesthe second volume 1125 of the second reservoir 1120 from the firstvolume 1115 of the first reservoir 1110. The second volume 1125 can beremoved from the second reservoir 1120 through the second externalopening 1140. The first reservoir 1110 is located above the secondreservoir 1120 when the unitary sampling device 1100 is in an uprightposition.

In some embodiments, the unitary sampling device 1100 can be utilizedwith a base or a clamp for supporting and/or positioning the unitarysampling device 1100 in an upright position. In some embodiments, thebase and/or clamp can be used to hold the unitary sampling device 1100in a stationary position. Similarly, the base or clamp can be removedfrom the unitary sampling device 1100 for portability.

FIG. 12 illustrates a unitary sampling device 1200. Unitary samplingdevice 1200 comprises a first reservoir 1210, a second reservoir 1220,and a plunger 1230. The plunger 1230 having the seal 1260 can be movedto displace the second volume 1225 of the second reservoir 1220. Thedistal end 1255 of plunger 1230 can extend below the second reservoir1220 and the second external opening 1240.

The sampling devices illustrated in FIGS. 2-12 of the present disclosurecan be formed from a number of materials. Materials useful for formingthe sampling devices can include, for example, glass, polymericmaterials, composite materials, and the like. These devices can also beconstructed of more than one material. Some examples of polymericmaterials include polypropylene, polycarbonate, acrylics, polystyrene,polyolefin, high density polyethylene, high density polypropylene, andthe like. In some embodiments, devices can be formed from one or moremethods of fabrication including, for example, injection molding, blowmolding, and by other fabrication techniques.

Concentration agents suitable for mixing with samples comprising amicroorganism for providing microorganism bound compositions aredescribed. Concentration agents are generally particulate or dispersiblein the sample, and also concentrate microorganisms present in largevolume samples. Such concentration agents have been found effective forcapturing microorganisms. The term “concentration agent” generallyrefers to a material for concentrating a general population ofmicroorganisms present in a sample. Examples of concentration agentshave been described in PCT Publication Nos. WO2009/009188;WO2009/085357; WO2009/046081; WO2009/046183; and U.S. Patent ApplicationPublication No. 2007/0269341, each of which is incorporated herein byreference in its entirety.

Concentration or capture using concentration agents (e.g., captureagents), in some embodiments, can be selected to be nonspecific orspecific to any particular strain, species, or type of microorganism andtherefore provide for the concentration of a general population ofmicroorganisms in a sample. In some embodiments, specific strains ofmicroorganisms can be detected from among the captured microorganismpopulation using any known detection method with strain-specific probesor with strain-selective culture media. Thus, the concentration agentscan be used, for example, in the detection of microbial contaminants orpathogens (particularly water-borne and food-borne pathogens such asbacteria) in clinical, food, environmental, or other samples.

In carrying out the method of the present disclosure, the concentrationagents can be used in any form that is amenable to sample contact andmicroorganism capture (for example, in particulate form or applied to asupport such as a dipstick, film, filter, tube, well, plate, beads,membrane, or channel of a microfluidic device, or the like). Preferably,the concentration agents are used in particulate form, more preferablycomprising microparticles (preferably, microparticles having a particlesize in the range of about 1 micrometer (more preferably, about 2micrometers) to about 100 micrometers (more preferably, about 50micrometers; even more preferably, about 25 micrometers; mostpreferably, about 15 micrometers; where any lower limit can be pairedwith any upper limit of the range).

Concentration agents useful for carrying out the method of the presentdisclosure include particulate concentration agents that comprise metal,metal oxide microparticles, metal silicates, diatomaceous earth, surfacemodified diatomaceous earth, particles having functional groups,biomolecules, fragments of biomolecules, nanoparticles, and combinationsthereof.

Some examples of concentration agents include iron, silica, titania,zirconia and others useful for collecting and concentrating samples. Insome embodiments, gamma-FeO(OH) (also known as lepidocrocite) can beused as a concentration agent. Such concentration agents have been foundto be more effective than other iron-containing concentration agents incapturing gram-negative bacteria, which are the microorganisms ofgreatest concern in regard to food- and water-borne illnesses and humanbacterial infections. The concentration agents can further include (inaddition to gamma-FeO(OH)) other components (for example, boehmite(α-AlO(OH)), clays, iron oxides, and silicon oxides), but, preferably,such other components do not significantly interfere with the intimatecontact of the sample and the concentration agent when carrying out themethod of the present disclosure. Gamma-FeO(OH) is also commerciallyavailable (for example, from Alfa Aesar, A Johnson Matthey Company, WardHill, Mass., and from Sigma-Aldrich Corporation, St. Louis, Mo.).

In carrying out the method of the present disclosure, the concentrationagents can be used in particulate form, more preferably comprisingmicroparticles (preferably, microparticles having particle sizes(largest dimension) in the range of about 3 micrometers (morepreferably, about 5 micrometer; most preferably, about 10 micrometers)to about 100 micrometers (more preferably, about 80 micrometers; evenmore preferably, about 50 micrometers; most preferably, about 35micrometers; where any lower limit can be paired with any upper limit ofthe range). Preferably, the particles are agglomerates of smallerparticles. The particles preferably comprise crystallites that are lessthan about 1 micrometer in size (preferably, less than about 0.5micrometer in size). The crystallites can be present as acicularcrystallites, as raft-like structures comprising acicular crystallites,or as combinations of the acicular crystallites and raft-likestructures. The concentration agents preferably have a surface area asmeasured by the BET (Brunauer-Emmett-Teller) method (calculation of thesurface area of solids by physical adsorption of nitrogen gas molecules)that is greater than about 25 square meters per gram (m²/g), morepreferably greater than about 50 m²/g. and most preferably greater thanabout 75 m²/g.

The preferred agglomerated form of such particles can provide adsorptivecapabilities of fine particle systems without the handling and otherhazards often associated with fine particles. In addition, suchagglomerate particles can settle readily in fluid and thus can providerapid separation of microorganisms from a fluid phase (as well asallowing low back pressure if used in filtration applications).

In some embodiments, metal silicates can be used as concentrationagents. Particularly useful metal silicates with a surface compositionhaving a metal atom to silicon atom ratio of less than or equal to about0.5, as determined by X-ray photoelectron spectroscopy (XPS).Preferably, the surface composition also comprises at least about 10average atomic percent carbon, as determined by X-ray photoelectronspectroscopy (XPS). XPS is a technique that can determine the elementalcomposition of the outermost approximately 3 to 10 nanometers (nm) of asample surface and that is sensitive to all elements in the periodictable except hydrogen and helium. XPS is a quantitative technique withdetection limits for most elements in the 0.1 to 1 atomic percentconcentration range. Preferred surface composition assessment conditionsfor XPS can include a take-off angle of 90 degrees measured with respectto the sample surface with a solid angle of acceptance of ±10 degrees.

When dispersed or suspended in water systems, metal silicates canexhibit surface charges that are characteristic of the material and thepH of the water system. The potential across the material-waterinterface is called the “zeta potential,” which can be calculated fromelectrophoretic mobilities (that is, from the rates at which theparticles of material travel between charged electrodes placed in thewater system). The concentration agents used in carrying out the methodof the present disclosure have zeta potentials that are more negativethan that of, for example, a common metal silicate such as ordinarytalc. Yet the concentration agents are more effective than talc inconcentrating microorganisms such as bacteria, the surfaces of whichgenerally tend to be negatively charged. Preferably, the concentrationagents have a negative zeta potential at a pH of about 7 (morepreferably, a Smoluchowski zeta potential in the range of about −9millivolts to about −25 millivolts at a pH of about 7; even morepreferably, a Smoluchowski zeta potential in the range of about −10millivolts to about −20 millivolts at a pH of about 7; most preferably,a Smoluchowski zeta potential in the range of about −11 millivolts toabout −15 millivolts at a pH of about 7).

Examples of useful metal silicates include amorphous silicates of metalssuch as magnesium, calcium, zinc, aluminum, iron, titanium, and the like(preferably, magnesium, zinc, iron, and titanium; more preferably,magnesium), and combinations thereof. Preferred are amorphous metalsilicates in at least partially fused particulate form (more preferably,amorphous, spheroidized metal silicates; most preferably, amorphous,spheroidized magnesium silicate). Metal silicates are known and can bechemically synthesized by known methods or obtained through the miningand processing of raw ores that are naturally-occurring.

Amorphous, at least partially fused particulate forms of metal silicatescan be prepared by any of the known methods of melting or softeningrelatively small feed particles (for example, average particle sizes upto about 25 microns) under controlled conditions to make generallyellipsoidal or spheroidal particles (that is, particles having magnifiedtwo-dimensional images that are generally rounded and free of sharpcorners or edges, including truly or substantially circular andelliptical shapes and any other rounded or curved shapes). Such methodsinclude atomization, fire polishing, direct fusion, and the like. Apreferred method is flame fusion, in which at least partially fused,substantially glassy particles are formed by direct fusion or firepolishing of solid feed particles (for example, as in the methoddescribed in U.S. Pat. No. 6,045,913 (Castle), the description of whichis incorporated herein by reference). Most preferably, such methods canbe utilized to produce amorphous, spheroidized metal silicates byconverting a substantial portion of irregularly-shaped feed particles(for example, from about 15 to about 99 volume percent; preferably, fromabout 50 to about 99 volume percent; more preferably, from about 75 toabout 99 volume percent; most preferably, from about 90 to about 99volume percent) to generally ellipsoidal or spheroidal particles.

Some amorphous metal silicates are commercially available. For example,amorphous, spheroidized magnesium silicate is commercially available foruse in cosmetic formulations (for example, as 3M Cosmetic MicrospheresCM-111, available from 3M Company, St. Paul, Minn.).

In some embodiments, amorphous metal silicates can further compriseother materials including oxides of metals (for example, iron ortitanium), crystalline metal silicates, other crystalline materials, andthe like, provided that the concentration agents have theabove-described surface compositions. The concentration agents, however,preferably contain essentially no crystalline silica.

In some embodiments, diatomaceous earth bearing, on at least a portionof its surface, a surface treatment comprising a surface modifiercomprising titanium dioxide, fine-nanoscale gold or platinum, or acombination thereof for use as concentration agents.

Thus, concentration agents comprising certain types of surface-treatedor surface-modified diatomaceous earth (namely, bearing a surfacetreatment comprising a surface modifier comprising titanium dioxide,fine-nanoscale gold or platinum, or a combination thereof) can beeffective when compared to untreated diatomaceous earth forconcentrating microorganisms. The surface treatment preferably furthercomprises a metal oxide selected from ferric oxide, zinc oxide, aluminumoxide, and the like, and combinations thereof (more preferably, ferricoxide). Although noble metals such as gold have been known to exhibitantimicrobial characteristics, the gold-containing concentration agentsused in the process of the invention surprisingly can be effective notonly in concentrating the microorganisms but also in leaving them viablefor purposes of detection or assay.

Useful surface modifiers include fine-nanoscale gold; fine-nanoscaleplatinum; fine-nanoscale gold in combination with at least one metaloxide (preferably, titanium dioxide, ferric oxide, or a combinationthereof); titanium dioxide; titanium dioxide in combination with atleast one other (that is, other than titanium dioxide) metal oxide; andthe like; and combinations thereof. Preferred surface modifiers includefine-nanoscale gold; fine-nanoscale platinum; fine-nanoscale gold incombination with at least ferric oxide or titanium dioxide; titaniumdioxide; titanium dioxide in combination with at least ferric oxide; andcombinations thereof.

More preferred surface modifiers include fine-nanoscale gold;fine-nanoscale platinum; fine-nanoscale gold in combination with ferricoxide or titanium dioxide; titanium dioxide; titanium dioxide incombination with ferric oxide; and combinations thereof (even morepreferably, fine-nanoscale gold; fine-nanoscale gold in combination withferric oxide or titanium dioxide; titanium dioxide in combination withferric oxide; and combinations thereof). Fine-nanoscale gold,fine-nanoscale gold in combination with ferric oxide or titaniumdioxide, and combinations thereof are most preferred.

In some embodiments, useful concentration agents (e.g., dispersibleparticles) can have binding groups bound to such particles. The binding(e.g., functional) groups of the particles can have a specific affinityfor specific microorganisms present in the samples. In some embodiments,the binding groups having more than site available for attachingmultiple microorganisms present in a sample.

In some embodiments, the particles have magnetic properties. Theparticles can, for example, have magnetic cores. Microorganism boundcompositions containing such concentration agents can be collected bythe application of a magnetic field, for example, to transfer thecomposition from the first reservoir to the second reservoir of asampling device.

In some embodiments, biomolecules (e.g., antibodies) can be covalentlybonded to particles by any of a variety of methods for formingconcentration agents. For example, glutaraldehyde, aldehyde-Schiff base,n-hydroxyl succinimide, azlactone, cyanogen bromide, maleic anhydride,etc., may be used as suitable attachment chemistries.

The biomolecule-binding group can be functionalized with variouschemical groups that allow for binding to a biomolecule. Such groups aretypically provided by biomolecule-binding compounds represented by theformula A-L-B. The biomolecule-binding group B may be any usefulfunctional group capable of reacting and forming a covalent bond(preferably a nonreversible covalent bond) with any of the biomoleculesof interest. A wide variety of such groups is known and may be useful.Generally the group B will be different from the group A(surface-bonding group). In this representation, L can be a bond or anyof a variety of organic linkers. Organic linkers L can be linear orbranched alkylene, arylene, or a combination of alkylene and arylenegroups, optionally including heteroatoms. For certain embodiments, the Lgroups do not include divalent alkylene oxide-containing oligomeric orpolymeric groups. For certain embodiments, if the L groups do includedivalent alkylene oxide-containing oligomeric or polymeric groups thatcould provide shielding and/or water-dispersible characteristics to thenanoparticles, they are not the only shielding and/or water-dispersiblegroups present on the nanoparticles.

Nonlimiting examples of such reactive groups B include those selectedfrom the group consisting of amines (particularly primary amines,although secondary amines can also be used, which can be aromatic and/oraliphatic), hydrazines, hydroxyl groups (—OH), sulfones, aldehydes,alcohols (—OR), oxiranes (such as ethylene oxides), halides (Cl, Br, I,F), N-oxysuccinimides, acrylates, acrylamides, alpha,beta-ethylenicallyor acetylenically unsaturated groups with electron withdrawing groups(e.g., alpha,beta-unsaturated ketones), carboxylates, esters,anhydrides, carbonates, oxalates, aziridines, epoxy groups,N-substituted maleimides, azlatones, and combinations thereof.

Examples of certain of these B groups with L linkers attached are shownbelow, wherein the B groups include aldehyde and hydroxyl groups,halides, esters, hydrazines (aliphatic or aromatic), andN-oxysuccinimides:

In some embodiments, vinyl sulfones, epoxy groups, acrylates, and aminesare preferred as they allow for direct attachment without complicatedreaction chemistry (as is needed with, for example, carboxylates). Thefollowing are representations of preferred B groups with L linkers,wherein the B groups include vinyl sulfone, epoxy, acrylate, and aminegroups:

Various combinations of the biomolecule-binding groups can be used. Theycan be on the same particle or on different particles.

In some embodiments, biomolecule-binding groups are those that arehydrolysis resistant. Hydrolysis resistant functional groups forreaction with biomolecules include acrylates, alpha,beta-unsaturatedketones, a N-sulfonyldicarboximide derivative, an acylsulfonamide, aN-sulfonylaminocarbonyl, a fluorinated ester, a cyclic azlactone, asulfonyl fluoride, a cyclic oxo-carbon acid (deltic, squaric, croconicand rhodizonic), a cyanuric fluoride, a vinyl sulfone, a perfluorinatedphenol, and various combinations thereof.

For biomolecule-binding compounds A-L-B, the surface-bonding groups Aare typically silanols, alkoxysilanes, or chlorosilanes, which can bemonofunctional, difunctional, or trifunctional. For example, the silanolgroups on the surfaces of the silica nanoparticles are reacted with atleast one silanol, alkoxysilane, or chlorosilane group of abiomolecule-binding compound to form a functionalized nanoparticle.

For certain embodiments, the biomolecule-binding groups includealpha,beta-ethylenically or acetylenically unsaturated group with anelectron withdrawing group. Nonlimiting examples of electron withdrawinggroups include carbonyls, ketones, esters, amides, —SO₂—, —SO—, —CO—CO—,—CO—COOR, sulfonamides, halides, trifluoromethyl, sulfonamides, halides,maleimides, maleates, or combinations thereof. For certain embodiments,the electron withdrawing groups is a ketone, ester, or amide.

The biomolecule-binding groups can be provided by biomolecule-bindingcompounds represented by the formula A-L-B. The biomolecule-bindinggroup B is an alpha,beta-ethylenically or acetylenically unsaturatedgroup. Generally, the group B will be different from the group A(surface-bonding group). In this representation, L can be a bond or anyof a variety of organic linkers, such that certain preferred group L-B(or simply B) has the following structures:

or

In certain embodiments the biomolecule-binding group is an acrylate oran alpha,beta-unsaturated ketone. Acrylates and alpha,beta-unsaturatedketones exhibit the desirable properties of stability in water over awide range of pH and yet also exhibit high reactivity with primaryamines to irreversibly form a Michael addition adduct.

A Michael addition adduct can result when anamino-group-bearing-biomolecule covalently bonds to abiomolecule-binding group by means of a carbon-nitrogen bond involvingan amino group of the biomolecule and the beta position of analpha,beta-ethylenically unsaturated group bearing a carbonyl unit atalpha position.

In some embodiments, acrylates and alpha,beta-unsaturated ketones can beused since they are compatible with a wide variety of surface-bondinggroups. In certain embodiments, the acrylate is multifunctional.Examples of biomolecule-binding compounds includeN-(3-acryloxy-2-hydroxypropyl) 3-aminopropyl triethoxysilane,3-acryloxypropyl trimethoxysilane, vinyl sulfone triethoxysilane-2, 1,1, 2-trifluorovinyl, 1,1,2-trichlorovinyl, 1,1-dichlorovinyl,1,1-difluorovinyl, 1-fluoro or 1-chlorovinyl silanes,alpha,beta-unsaturated containing silanes, silane-containing quinones.Those of ordinary skill in the art will recognize that a wide variety ofother biomolecule-binding compounds are useful in the present disclosureas compounds that can be used to functionalize particles withbiomolecule-binding groups. Preferably, a sufficient amount ofbiomolecule-binding compound is reacted on the surface of such particlesto provide the desired level of attachment of biomolecule of interest (apolypeptide such as an antibody, preferably an IgG antibody).

In some embodiments, the biomolecule-binding group can include an amineand/or a hydrazine. The amine and/or hydrazine may be aromatic,aliphatic, or a combination thereof. The amine may be primary orsecondary, although it is preferably a primary amine, the more preferredprimary amines are hydrophilic amines including poly(ethylene oxide)amines and polyimines.

In some embodiments, the biomolecule-binding group can include an arylamine and/or an aryl hydrazine. The amine may be primary or secondary(i.e., nontertiary), although it is preferably a primary amine. In suchembodiments, the biomolecule-binding groups can be provided bybiomolecule-binding compounds represented by the formula A-L-B, whereinthe biomolecule-binding group B is an aryl nontertiary amine and/or arylhydrazine group. Generally, the group B will be different from the groupA (surface-bonding group). In this representation, L can be a bond orany of a variety of organic linkers, such that certain preferred groupsL-B (or simply B) have the following structures:

In some embodiments, the B groups can include an aryl amine and/or arylhydrazine and reacts with a biomolecule having a free carbonyl groupthrough a Schiff base mechanism, thereby forming a linkage of theformula —Ar—N═C(H)-biomolecule, or —Ar—NHN═C(H)-biomolecule wherein Aris an aryl group, which may be unsubstituted or substituted. The arylgroup may include a single aromatic ring or multiple aromatic rings,which may or may not include heteroatoms (particularly, S, N, O).Examples include naphthalene, anthracene, pyrene, and biphenyl. If thearyl group is substituted, the substituents (e.g., hydroxyl, carboxyl,methoxy, methyl, amino groups) should not interfere sterically orelectronically with the function of the aryl amine and/or aryl hydrazineas the biomolecule-binding group.

The size of the aryl group should be balanced against the number andtype of water-dispersible groups to avoid excessive agglomeration of thenanoparticles. If desired, the aryl group can be substituted withhydrophilic groups to assist in the dispersion of the particles.

In some embodiments, examples of biomolecule-binding compounds (i.e.,compounds capable of providing a biomolecule-binding group having anaryl amine and/or aryl hydrazine group), represented by the formulaA-L-B, can include 4-aminophenyltrimethoxy silane.

Preferably, a sufficient amount of biomolecule-binding compound can bereacted with the particles so as to provide the desired level ofattachment of biomolecule of interest (e.g., an oxidized polypeptidesuch as an oxidized antibody, preferably an IgG antibody).

In some embodiments, biomolecule-binding groups can include primaryaliphatic and/or aromatic amines, and the biomolecule-binding groupshaving a biomolecule covalently bonded thereto include abiotin-containing group covalently bonded to the surface of theparticles through the amine-functionalized groups.

In some embodiments, amine-containing biomolecule-binding groups can bearomatic amines. As aliphatic amines, they generally have no less thanabout 6 carbon atoms, particularly when the water-dispersible and/orshielding groups include poly(alkylene oxide)-containing groups.

In some embodiments, biomolecules useful as concentration agents can beany chemical compound that naturally occurs in living organisms, as wellas derivatives or fragments of such naturally occurring compounds.Biomolecules consist primarily of carbon and hydrogen, along withnitrogen, oxygen, phosphorus, and sulfur. Other elements sometimes areincorporated but are much less common. Biomolecules include, but are notlimited to, proteins, antibodies, polypeptides, carbohydrates,polysaccharides, lipids, fatty acids, steroids, prostaglandins,prostacyclines, vitamins, cofactors, cytokines, and nucleic acids(including DNA, RNA, nucleosides, nucleotides, purines, andpyrimidines), metabolic products that are produced by living organismsincluding, for example, antibiotics and toxins. Biomolecules may alsoinclude derivatives of naturally occurring biomolecules, such as aprotein or antibody that has been modified with chemicals (e.g.,oxidized with sodium periodate). Biomolecules may also includecrosslinked naturally occurring biomolecules, or a crosslinked productof a naturally occurring biomolecule with a chemical substance. Thus, asused herein, the term “biomolecule” includes, but is not limited to,both unmodified and modified molecules (e.g., glycosylated proteins,oxidized antibodies) and fragments thereof (e.g., protein fragments).Fragments of biomolecules can include those resulting from hydrolysisdue to chemical, enzymatic, or irradiation treatments, for example.

In certain embodiments, biomolecules may be covalently bonded to one ormore of the biomolecule-binding groups. In certain embodiments, thebiomolecule includes or can be modified to include an aldehyde groupprior to its attachment to the biomolecule-binding group.

The attachment of an antibody (e.g., oxidized antibody) or otherbiomolecule (e.g., oxidized biomolecule) typically takes place undermild conditions, and can occur under a broad pH range, preferably pH at4-11, more preferably pH at 6-10, and most preferably pH at 7-9. Thepreferred temperature for attachment of an antibody (e.g., oxidizedantibody) or other biomolecule (e.g., oxidized biomolecule) is roomtemperature. Also, lower or higher temperatures can be used, but not attemperatures which denature the biomolecule. This chemistry is suitablefor all kinds of biological media, basic and even mildly acidic buffersolutions, and in mixed solvents including solvents such as DMSO oracetonitrile.

In some embodiments, biomolecules such as biotin can be used to capturetarget biomolecular analytes (e.g., antibodies). Such amine-containinggroups with biotin bonded thereto can be formed by the reaction of(+)-Biotin-N-hydroxy-succinimide ester compounds with a primaryaliphatic and/or aromatic amine (the biomolecule-binding group), whereinthe amine functional group is bonded to a surface through linking groupL. Alternatively, the reaction of (+)-Biotin-N-hydroxy-succinimide estercompounds with the amine can be carried out prior to binding to thesurface of the silica nanoparticles.

Biotin, also known as vitamin H orcis-hexahydro-2-oxo-1H-thieno-[3-,4]-imidazole-4-pentanoic acid, is abasic vitamin which is essential for most organisms including bacteriaand yeast. Biotin has a molecular weight of 244 daltons, much lower thanits binding partners, avidin and streptavidin. Biotin is also an enzymecofactor of pyruvate carboxylase, trans-carboxylase,acetyl-CoA-carboxylase and beta-methylcrotonyl-CoA carboxylase whichtogether carboxylate a wide variety of substrates. Derivatives ofbiotin, such as N-hydroxysuccinimide esters of biotin (referred to asNHS-biotin), N-hydroxysulfosuccinimide esters of biotin (referred to assulfo-NHS-biotin), sulfosuccinimidyl-6-[biotinamido]hexanoate (referredto as sulfo-NHS-LC-biotin),sulfosuccinimidyl-6-[biotinamido]-6-hexanamidohexanoate (referred to assulfo-NHS-LC-LC-biotin), and N-hydroxysuccinimide PEG₁₂-biotins orN-hydroxysuccinimide PEG₄-biotins (referred to as NHS-PEO₁₂-biotin orsulfo-NHS-PEO₄-biotin), can be used to attach to amines on silicananoparticles. Thus, using this nomenclature, the biotin or biotinderivatives are the biomolecules, whereas the biomolecule-binding groupsare the amines. The biotin-containing compound (e.g., biotin orderivatives of biotin) forms a bond with avidin or strepavidin, thecomplex of which is capable of binding to an antibody, which can be thetarget analyte or can be specific for a target analyte (e.g., abacterium).

The selective attachment of a target biological analyte can be achieveddirectly or it may be achieved through a capture agent, e.g.,antigen-antibody binding (where the target biological analyte itselfincludes the antigen bound to an antibody immobilized on the detectionsurface).

Concentration agents having capture agents can include species (e.g.,molecules, groups of molecules) that have high affinity for a targetbiological analyte, and preferably are specific for a target analyte.Capture agents include, for example, antibodies and fragments thereof(Fab, Fab′, Fc), polypeptides, aptamers, DNA, RNA, oligonucleotides,proteins, antibodies, carbohydrates, polysaccharides, lipids, fattyacids, steroids, vitamins, cytokines, lectins, cofactors, and receptors(e.g., phage receptors). Capture agents may also include derivatives ofnaturally occurring biomolecules, such as a protein or antibody that hasbeen modified with chemicals. These may also include crosslinkednaturally occurring biomolecules, or a crosslinked product of anaturally occurring biomolecule with a chemical substance.

Some biomolecule capture agents suitable for use in the presentdisclosure include polypeptides including antibodies, antibodyconjugates, and proteins such as avidin, streptavidin, and clumpingfactor). In particular, biomolecule capture agents are antibodies. Theterm “antibody” is intended to include whole antibodies of any isotype(IgG, IgA, IgM, IgE, etc.), and fragments thereof from vertebrate, e.g.,mammalian species, which are also specifically reactive with foreigncompounds, e.g., proteins.

The antibodies can be monoclonal, polyclonal, or combinations thereof.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as whole antibodies.Thus, the term includes segments of proteolytically cleaved orrecombinantly prepared portions of an antibody molecule that are capableof selectively reacting with a certain protein. Nonlimiting examples ofsuch proteolytic and/or recombinant fragments include Fab, F(ab′)₂, Fv,and single chain antibodies (scFv) containing a VL and/or VH domainjoined by a peptide linker. The scFv's can be covalently ornon-covalently linked to form antibodies having two or more bindingsites. Antibodies can be labeled with any detectable moieties known toone skilled in the art. In some aspects, the antibody that binds to ananalyte one wishes to measure (the primary antibody) is not labeled, butis instead detected indirectly by binding of a labeled secondaryantibody or other reagent that specifically binds to the primaryantibody.

The surface coverage and packing of the capture agent on the surface ofthe concentration agent may affect the sensitivity of detecting thetarget biological analyte. The immobilization chemistry that links thecapture agent to the surface may play a role in the packing of thecapture agents, preserving the activity of the capture agent, and mayalso contribute to the reproducibility and shelf-life of the surfaces. Avariety of immobilization methods described elsewhere herein may be usedin connection with surfaces to achieve the goals of high yield,activity, shelf-life, and stability.

Bioaffinity pairs, such as antigen/hapten, antibody/antigen bindingfragment of the antibody, or complementary nucleic acids,bioreceptor/ligand (interleukin-4 and its receptor) may be used toattach capture agents. One of the pairs of such biomolecules iscovalently attached to the biomolecule-binding agent. These biomoleculesform part of a “capture agent” for a target biological analyte. Forexample, the strong bond formed between biotin and avidin and/orstreptavidin may be particularly useful when attaching an antibody to asurface. Preferably, streptavidin can be used as a means to attach anantibody, to a surface. Streptavidin is a tetrameric protein isolatedfrom Streptomyces avidinii that binds tightly to the vitamin biotin.Proteins, such as streptavidin, can be attached to surfaces through anumber of chemistries.

Derivatives of biotin, such as N-hydroxysuccinimide esters of biotin(referred to as NHS-biotin), N-hydroxysulfosuccinimide esters of biotin(referred to as sulfo-NHS-biotin),sulfosuccinimidyl-6-[biotinamido]hexanoate (referred to assulfo-NHS-LC-biotin),sulfosuccinimidyl-6-[biotinamido]-6-hexanamidohexanoate (referred to assulfo-NHS-LC-LC-biotin), and N-hydroxysuccinimide PEG₁₂-biotins, andN-hydroxysuccinimide PEG₄-biotins (referred to as NHS-PEO₁₂-biotin orsulfo-NHS-PEO₄-biotin), can be used to attach biotins to biomolecules,such as antibodies, at primary amino acid groups. These biotinylatedbiomolecules can subsequently be attached to a surface that hasstreptavidin attached thereto.

“Target biological analytes” include, for example, tissues, cells, orbiomolecules therewithin or derived therefrom (e.g., organism-specificantigens, enzymes, or other proteins, peptides, carbohydrates, toxins,or prions, cell wall components or fragments, flagella, pili, nucleicacids, antibodies).

As used herein, the term “tissue” refers to multicellular aggregates oran organ derived from animals or plants, and includes both viable andnonviable cells, connective tissue, and interstitial fluids. “Cell”refers to the basic structural and functional unit of all livingorganisms, including animals, plants, and single-celled microorganisms.

Concentration agents useful in the method of the present disclosure canbe applied to a variety of different types of samples comprisingmicroorganisms. Samples having low microorganism concentrations can havemicroorganisms within the sample concentrated as escribed herein. Someexamples of samples can include, but not limited to, medical,environmental, food, feed, clinical, and laboratory samples, andcombinations thereof. Medical or veterinary samples can include, forexample, cells, tissues, or fluids from a biological source (forexample, a human or an animal) that are to be assayed for clinicaldiagnosis. Environmental samples can be, for example, from a medical orveterinary facility, an industrial facility, soil, a water source, afood preparation area (food contact and non-contact areas),environmental surfaces (e.g., floors, walls, ceilings, fomites,equipment, water, water containers, and air filters), a laboratory, oran area that has been potentially subjected to bioterrorism. Foodprocessing, handling, and preparation area samples, potable water andenvironmental surfaces are preferred, as these are often of particularconcern in regard to food supply contamination by bacterial pathogens.

A sample useful in the method of the present disclosure can be in theform of a fluid (e.g., a liquid, or a dispersion or suspension). In someembodiments, samples obtained in the form of a liquid can beconcentrated with a dispersible concentration agent so thatmicroorganism bound composition can be formed.

Examples of samples that can be used (either directly or after treatmentto provide a fluid sample) in carrying out the process of the inventioninclude foods (for example, fresh produce rinsates or ready-to-eat lunchor “deli” meats), beverages (for example, juices or carbonatedbeverages), potable water, and biological fluids (for example, wholeblood or a component thereof such as plasma, a platelet-enriched bloodfraction, a platelet concentrate, or packed red blood cells); cellpreparations (e.g., dispersed tissue, bone marrow aspirates, orvertebral body bone marrow); cell suspensions; urine, saliva, and otherbody fluids; bone marrow; spinal fluid; and the like, as well as lysedpreparations, such as cell lysates, which can be formed using knownprocedures such as the use of lysing buffers, and the like. Preferredsamples include foods, beverages, potable water, biological fluids,environmental samples, and combinations thereof (with foods, beverages,potable water, environmental samples, and combinations thereof beingmore preferred).

Sample volume of the sampling device can vary, depending upon theparticular application. For example, when the method of the presentdisclosure is used for clinical diagnostic or research application, thevolume of the sample can typically be in a range from about 0.5milliliters to about 10 milliliters. When the method is used for foodpathogen testing assay or for potable water safety testing, the volumeof the sample can typically be in the milliliter to liter range (forexample, 100 milliliters to 3 liters). In an industrial application,such as bio-processing or pharmaceutical formulation, the sample volumecan be tens of thousands of liters.

The process of the invention can isolate microorganisms from a sample ina concentrated state using a concentration agent, and can also allow theisolation of low levels of microorganisms from sample matrix componentsthat can inhibit detection procedures that are to be used. In all ofthese cases, the process of the invention can be used in addition to, orin replacement of, other methods of microorganism concentration. Thus,optionally, cultures can be grown from samples either before or aftercarrying out the process of the present disclosure, if additionalconcentration is desired.

A variety of samples comprising microorganisms can be concentrated asmicroorganism bound compositions, and collected (e.g., transferred) byone of the methods described herein using the sampling devices of thepresent disclosure. Microorganisms present in the samples for providingthe microorganism bound compositions can include, for example, fungi,yeasts, protozoans, viruses, and the like, and combinations thereof. Insome embodiments, the microorganisms can include gram-negative bacteria,gram-positive bacteria, and combinations thereof. The method ofconcentrating microorganisms using the sampling devices described hereinhas utility for concentrating and detecting pathogens, which can beimportant for food safety or for medical, environmental, oranti-terrorism reasons. The process can be particularly useful in thedetection of pathogenic bacteria (for example, both gram negative andgram positive bacteria), as well as various yeasts, molds, andmycoplasmas (and combinations of any of these). As used herein, the term“microorganism” refers to prokaryotic or eukaryotic organisms that aregenerally classified as bacteria, viruses, yeast, filamentous fungi, andprotozoa. As used herein, the term “prokaryotic organism” includes allforms of microorganisms considered to be bacteria including cocci,bacilli, spirochetes, sheroplasts, protoplasts, spores, etc.

Microbes (i.e., microorganisms) of particular interest include Grampositive bacteria, Gram negative bacteria, fungi, protozoa, mycoplasma,yeast, viruses. Particularly relevant organisms include members of thefamily Enterobacteriaceae, or genera Staphylococcus spp., Streptococcusspp., Pseudomonas spp., Clostridium spp., Enterococcus spp., Esherichiaspp., Bacillus spp., Listeria spp., Legionella spp., Vibrio spp., aswell as herpes virus, Aspergillus spp., Fusarium spp., and Candida spp.Particularly virulent organisms include Staphylococcus aureus (includingresistant strains such as Methicillin Resistant Staphylococcus aureus(MRSA), Vancomycin Resistant Staphylococcus aureus (VRSA), VancomycinIntermediate-resistant Staphylococcus aureus (VISA)), Clostridiumdifficile, S. epidermidis, Streptococcus pneumoniae, S. agalactiae, S.pyogenes, Enterococcus faecalis, Vancomycin Resistant Enterococcus(VRE), Bacillus anthracis, Bacillus amyloliquefaciens, Bacillusamylolyticus, Bacillus cereus, Bacillus coagulans, Bacillus macerans,Bacillus megaterium, Bacillus polymyxa, Bacillus stearothermophillus,Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Aspergillusniger, A. fumigatus, A. clavatus, Fusarium solani, F. oxysporum, F.chlamydosporum, Listeria monocytogenes, Vibrio cholera, Vparahemolyticus, Salmonella cholerasuis, S. typhi, S. typhimurium,Candida albicans, C. glabrata, C. krusei, Strep A, Strep B,Agrobacterium tumefaciens, Alcaligenes xylosoxydans subsp.denitrificans, Sphingomonas paucimobilis, and multiple drug resistantGram negative rods (MDR).

Such microbes or other species of interest can be analyzed in a testsample that may be derived from any source, such as a physiologicalfluid, e.g., blood, saliva, ocular lens fluid, synovial fluid, cerebralspinal fluid, pus, sweat, exudate, urine, mucous, lactation milk, or thelike. Further, the test sample may be derived from a body site, e.g.,wound, skin, nares, scalp, nails, etc.

Besides physiological fluids, other test samples may include otherliquids as well as solid(s) dissolved in a liquid medium. Samples ofinterest may include process streams, water, soil, plants or othervegetation, air, surfaces (e.g., contaminated surfaces), and the like.

In some embodiments, methods for concentrating microorganisms can becarried out by any of various known or hereafter developed methods forproviding contact and/or mixing between two materials, for example, informing a dispersion. For example, the concentration agent (e.g., as aparticulate) can be added to the sample comprising a microorganism, orthe sample can be added to the concentration agent within the samplingdevice. The concentration agent and the sample can be combined in any ofa variety of sampling devices described within the present application.Optionally, but preferably, the sampling device is capped, closed, orsealed prior to use so as to reduce contamination prior to the additionof the sample. Similarly, the sampling device used herein is capable ofbeing capped, closed, or sealed after use for containment, storage, andease of portability.

Suitable sampling devices (e.g., unitary sampling devices or dualcomponent sampling devices) for carrying out the method of the presentdisclosure can be determined by the size of the sample. Useful samplingdevices can vary widely in size and design. In some embodiments,sampling devices can be small having a volume of 0.5 milliliter, orlarger, such that the volume of the sampling device can be in a range of100 milliliters to 3 liters. In some embodiments, the sampling deviceand the concentration agent that comes into contact with the sample canbe sterilized (for example, by controlled heat, ethylene oxide gas,hydrogen peroxide, or radiation) prior to use, in order to reduce orprevent any contamination of the sample that might cause detectionerrors. The amount of concentration agent that is sufficient to captureor concentrate the microorganisms of a particular sample for successfuldetection will vary (depending upon, for example, the nature and form ofthe concentration agent and sample volume) and can be readily determinedby one skilled in the art. The capture efficiency (the percent ofmicroorganisms in the sample bound to concentration agent) can generallybe increased by allowing increased time for the microorganism to come incontact with the concentration agent. The capture efficiency can also beincreased by having a higher concentration of concentration agent, whichdecreases the mean diffusion distance a microorganism must travel to becaptured, leading to a shorter incubation time. Therefore, as agenerality, the more concentration agent added, the shorter incubationtime necessary to capture the same amount of microorganisms. In someembodiments, for example, 10 milligrams of concentration agent for everymilliliter of sample can be useful for some applications. For otherapplications, concentration agents in the range of 1-50 milligrams permilliliter of sample can be utilized.

If desired, contacting or mixing within the sampling device can beeffected by passing concentration agents at least once through a sample(for example, by relying upon gravitational settling or by other methodsover a period of time. Contact can be enhanced by mixing (for example,by stirring, shaking, or use of a rocking platform) such that theparticles of concentration agent repeatedly pass or settle through asubstantial portion of the sample. For small volumes on the order ofmicroliters (typically less than 10 milliliters), mixing can be rapidsuch as by vortexing or “nutation,” for example as described in U.S.Pat. No. 5,238,812 (Coulter et al.), the description of which isincorporated herein by reference. For larger volumes on the order ofgreater than or equal to 1 milliliter (typically 1 milliliter to 3liters), mixing can be achieved by gently tumbling the concentrationagent and the sample in an “end over end” fashion, for example asdescribed in U.S. Pat. No. 5,576,185 (Coulter et al.), the descriptionof which is incorporated herein by reference. Contacting can be carriedout for a desired period (for example, for sample volumes of about 100milliliters or less, up to about 60 minutes of contacting can be useful;preferably, about 15 seconds to about 10 minutes or longer; morepreferably, about 15 seconds to about 5 minutes).

Thus, in carrying out the method of the present disclosure, mixing (forexample, agitation, rocking, or stirring) and optionally incubation canbe used in order to increase microorganism contact with theconcentration agent. A preferred contacting method includes both mixing(for example, for about 15 seconds to about 5 minutes) and incubating(for example, for about 3 minutes to about 4 hours) a microorganismcontaining samples with concentration agent. If desired, one or moreadditives (for example, lysis reagents, nucleic acid capture reagents(for example, magnetic beads), microbial growth media, buffers (forexample, to disperse or extract a solid sample), microbial stainingreagents, washing buffers (for example, to wash away unbound material),elution agents (for example, serum albumin), surfactants (for example,Triton X-100 nonionic surfactant available from Union Carbide Chemicalsand Plastics, Houston, Tex.), mechanical abrasion/elution agents (forexample, glass beads), and the like) can be added to the combination ofconcentration agent and sample.

The microorganism bound composition formed herein can be collected inthe second reservoir of the sampling device. Preferably, collecting ofthe microorganism bound composition can be achieved by relying, at leastin part, upon gravitational settling (gravity sedimentation; forexample, over a time period of about 5 minutes to about 4 hours). Insome embodiments, however, it can be desirable to accelerate collection(for example, by centrifugation or filtration) or to use combinations ofany of the collection methods.

Microorganisms that have been captured or bound by the concentrationagent described herein forming microorganism bound compositions can bedetected by essentially any desired method that is currently known orhereafter developed. Such methods include, for example, culture-basedmethods (which can be preferred when time permits), microscopy (forexample, using a transmitted light microscope or an epifluorescencemicroscope, which can be used for visualizing microorganisms tagged withfluorescent dyes) and other imaging methods, immunological detectionmethods, and genetic detection methods. The detection process followingmicroorganism capture optionally can include washing to remove samplematrix components.

In some embodiments, microorganisms that have been captured or bound bythe concentration agent can also be removed from the devices, placed ona culture dish, allowed to grow for a certain period of time and thecolonies can be detected by use of bioluminescence reagents and imagingof the plate using an imaging system such as Milliflex® RapidMicrobiology Detection and Enumeration System (Millipore, Bedford,Mass.).

In some embodiments, the microorganism bound composition can betransferred to a suitable reaction cuvette/vessel and directly detectedby adding bioluminescent reagents to the vessel. Bioluminescence can bequantified by measuring bioluminescence in a luminometer.

In some applications, the microorganism bound composition within thesampling device can be transferred to a laboratory for detection. Forcertain applications, microorganism bound compositions can be removedand placed into a sterile container for transport to a laboratory fordetection where, the container can be designed to avoid loss ofviability of the microorganisms. Optionally, a preservative can be addedto the container to maintain the viability of the microorganisms duringtransport.

In some embodiments, the microorganism bound composition residing in thesecond reservoir can be analyzed for microorganisms. In someembodiments, the microorganism bound composition can be removed from thesecond reservoir and analyzed for microorganisms. Analytical techniquesuseful for detecting such microorganisms of the microorganism boundcomposition be accomplished, for example, colorimetrically,electrochemically, fluorimetrically, lumimetrically, by culturing, byutilizing an immunoassay, or by utilizing enzyme assays or throughgenetic analysis.

Sampling devices of the present disclosure provide for convenience andportability as equipped for concentrating low levels of microorganismsin the presence of concentration agents from large volume samples. Thesampling devices described herein are inexpensive and recyclable thuseliminating the need for cleaning and sterilizing these devices beforeand after each use. A known volume of the microorganism boundcomposition can be collected from the second reservoir and subsequentlyanalyzed for identifying microorganisms present in such samples.

The disclosure will be further clarified by the following non-limitingexamples which are exemplary and not intended to limit the scope of thedisclosure.

EXAMPLES

Unless otherwise noted, all parts, percentages, and ratios reported inthe following examples are on a weight basis, and all reagents used inthe examples were obtained, or are available, from the chemicalsuppliers described below, or can be synthesized by conventionaltechniques.

Example 1 Unitary Sampling Device

A unitary sampling device illustrated in FIG. 2 was prepared. A 250milliliter polypropylene centrifuge tube (#430776; Corning Inc., Ithaca,N.Y.) was utilized for the first reservoir. A circular opening (˜6 mm)was formed in the narrow end of the tube to form an angled opening A 50milliliter polypropylene centrifuge tube (#430921; Corning, Inc.,Ithaca, N.Y.) was used for the second reservoir. The 50 ml tube washeated to soften the polypropylene and the 50 ml tube was placedadjacent to the 250 ml tube to form the unitary sampling device of FIG.2.

Example 2 Dual Component Sampling Device

A dual component sampling device illustrated by FIG. 3 was prepared. A250 ml centrifuge tube (described above) was utilized for the firstreservoir with a 6 mm opening. A 2 mm threaded female luer lock wasinserted into the 6 mm opening. The luer lock was attached in positionwith structural adhesive. A 5 ml polypropylene syringe (detachableaspirable second reservoir) was attached to the luer lock to form thedual component sampling device of FIG. 3.

Example 3 Unitary Sampling Device

A unitary sampling device of FIG. 7 was prepared as described in Example1 with the exception that the 6 mm circular opening was formed notcentered on the axis.

Example 4 Unitary Sampling Device

A unitary sampling device of FIG. 2 was prepared as described in Example1 with the exception that the second reservoir was formed from a 100 mlcentrifuge tube.

Example 5 Unitary Sampling Device

A unitary sampling device of FIG. 2 was prepared as described in Example1 with the exception that the second reservoir was formed from a 25 mlcentrifuge tube.

Example 6 Unitary Sampling Device

A unitary sampling device of FIG. 2 as described in Example 1 was used.The resealable external opening of the second reservoir was equippedwith a closure having a protrusion as illustrated in FIG. 5.

Method for Concentrating Microorganisms Using Sampling Devices

An isolated E. coli (ATCC 51813) colony was inoculated from a streakplate into 5 ml BBL Trypticase Soy Broth (Becton Dickinson, Sparks, MD)and incubated at 37° C. for 18-20 hours (overnight culture). Theovernight culture at ˜10⁹ colony, having units/ml, was diluted inButterfield's Buffer (pH 7.2, VWR; West Chester, Pa.). A 1:1000 dilutionfrom a 10² bacteria/ml dilution was prepared in 100 ml of water with afinal concentration of 0.1/ml (10 cfus total). One milliliter of afilter sterilized, 100× concentrated adsorption buffer (pH 7.2containing 5 mM KCl, 1 mM CaCl₂, 0.1 mM MgCl₂ and 1 mM K₂HPO₄) wasadded.

A concentration agent, 3M microparticles (250 milligrams) (amorphous,spheroidized magnesium silicate X296-Talc; 3M Company, St. Paul, Minn.;U.S. Pat. No. 6,045,913)) was added to each of Examples 1-6 (samplingdevices). One hundred milliliters of E. coli spiked water was also addedto each of the sampling devices. The sampling devices were each sealedwith a closure and incubated at room temperature (25° C.) for 60 minuteson a Thermolyne VariMix rocking (mixing) platform at 14 cycles/minute(Barnstead International, Dubuque, Iowa). After the mixing andincubation steps, the sampling devices were not disturbed for 45-60minutes. Similarly, tubes containing E coli spiked water without theconcentration were treated to the same conditions.

In order to collect the microorganism bound composition, the samplingdevices (Examples 1, 3-6) were independently inverted so that thecomposition was collected in the second reservoir. The microorganismbound composition (2-5 milliliters) was pipetted from the secondreservoir and transferred to a 15 ml sterile polypropylene tube (VWR,West Chester, Pa.). The microorganism bound composition within theirrespective tubes was mixed, and pipetted lml at a time onto EC/CCPetrifilm (3M Company, St. Paul, Minn.).

In Example 2, the detachable aspirable second reservoir containing aplunger was drawn to acquire 3-4 ml of the microorganism boundcomposition to be added to the 15 ml sterile polypropylene tube, andfollowed by mixing and pipetting 1 ml at a time onto EC/CC Petrifilm.

The petrifilms of Examples 1-6 were processed per the manufacturer'sinstructions. E. coli/Coliform Count was quantified using a EC/CCPetrifilm Plate Reader (3M Company, St. Paul., Minn.). Results forExamples 1-6 were calculated with the formula listed below:Capture efficiency=(number of colonies from concentration agent onfilter/number of colonies in the plated untreated control (noconcentration agent))×100

Capture efficiency of E. Coli using the sampling devices of Examples 1-6is illustrated in Table 1. The capture data was obtained from EC/CCPetrifilm on which the retrieved microorganism bound composition wasplated.

TABLE 1 Capture of E coli from 100 ml water samples E. Coli in water E.Coli recovered from Capture sample concentration agent* EfficiencyExample (cfus) (cfus) (percent) 1 12 11 92 2 13 13 100 3 12 10 83 4 1315 115 5 13 8 62 6 11 11 100 *Microorganism bound composition

Various modifications and alterations of this disclosure will beapparent to those skilled in the art without departing from the scopeand spirit of this disclosure, and it should be understood that thisdisclosure is not limited to the illustrative elements set forth herein.

The invention claimed is:
 1. A method for concentrating microorganismscomprising the steps of: providing a sampling device comprising aunitary first compartment comprising: a first reservoir having a firstvolume; a second reservoir having a second volume at least oneresealable external opening; and a passageway connecting the firstreservoir to the second reservoir, the passageway having an opening thatprovides fluid communication between the first reservoir and the secondreservoir, wherein the entire first volume is above the opening when thesampling device is in an upright position, wherein the entire secondvolume is not above the opening when the sampling device is in anyposition; and a second component comprising a closure having aprotrusion, the closure coupled to the at least one resealable externalopening, wherein in a first position the protrusion permits fluid flowthrough the passageway and wherein in a second position the protrusionprevents fluid flow through the passageway; mixing a concentration agentand a sample comprising a microorganism in the unitary sampling deviceto provide a microorganism bound composition; and inverting the unitarysampling device such that the second reservoir is oriented substantiallyabove the first reservoir for collecting a major portion of themicroorganism bound composition from the second reservoir.
 2. The methodof claim 1, wherein providing a unitary sampling device comprisesproviding a unitary sampling device wherein a ratio of the entire volumeof the first reservoir to the entire volume of the second reservoir thatis in a range of about 10:1 to about 1000:1.
 3. The method of claim 1,wherein providing a unitary sampling device further comprises providinga unitary sampling device wherein the first reservoir has a resealableexternal opening.
 4. The method of claim 1, wherein providing a unitarysampling device comprises providing a unitary sampling device whereinthe passageway has a conical geometry.
 5. The method of claim 1, whereinproviding a unitary sampling device comprises providing a unitarysampling device wherein the second opening is centered on an axisextending from a center of the first reservoir to the second reservoir.6. The method of claim 1, wherein providing a unitary sampling devicecomprises providing a unitary sampling device wherein the second openingis not centered on an axis extending from a center of the firstreservoir to the second reservoir.
 7. The method of claim 1, whereinproviding a unitary sampling device further comprises providing aunitary sampling device wherein the resealable external opening has aclosure comprising a protrusion which partially occludes the secondopening of the passageway.
 8. The method claim 1, wherein mixing aconcentration agent and a sample comprising a microorganism in theunitary sampling device to provide a microorganism bound compositioncomprises mixing a concentration agent selected from the groupconsisting of particles with affinity ligands, particles withoutaffinity ligands, antibodies or antigen binding fragments, receptors andcombinations thereof.
 9. The method of claim 1, wherein mixing aconcentration agent and a sample comprising a microorganism in theunitary sampling device to provide a microorganism bound compositionfurther comprises mixing a concentration agent, a sample comprising amicroorganism, and a buffer in the unitary sampling device.
 10. Themethod of claim 1, wherein mixing a concentration agent and a samplecomprising a microorganism in the unitary sampling device to provide amicroorganism bound composition further comprises mixing a concentrationagent, a sample comprising a microorganism, a detection agent and agrowth medium in the unitary sampling device.